Dec 23, 03

 

Tissue homogenization in RIPA buffer

 

50mM HEPES pH 7.4

150mM NaCl

1% NP40

0.5% Na deoxycholate

0.1M PMSF (a proteinase inhibitor)

 

So, to prepare 50ml of RIPA buffer:

0.438g NaCl, 2.5ml of 1M HEPES, 0.5ml of NP40 (or its equivalent ), 0.25g  Na deoxycholate.

 

PMSF is very toxic and is only active within 60min of adding. Thus, do not add it to the buffer but add it in 1:100 dilution directly to the homogenized sample (just prior to starting).

 

 

(1) Homogenize in RIPA buffer

 

(2) Rotate end-over-end for 60min/4C

 

(3) Spin (low speed at 10,000 x g /10min), keep the supernatant.

 

(4) Take aliquote of the supernatant and add 4X SDS-buffer

 

(5) Boil 10min

 

(6) Load (8-14% gel).

 

 

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